In order to determine the presence or concentration of target ligands or analytes in a fluid, immunoassays utilize binding proteins, such as antibodies, to specifically bind to target ligands or analytes. Commonly, a signal is generated from the immunoassay to provide a detectable or sensible result. A class of useful signal generators are fluorescent molecules because of their high specific activity.
Fluorescent molecules have been utilized in immunoassays as signal generators in several ways. For example, enzymes can catalyze the production of fluorescent dyes from a substrate, such that the amount of fluorescent dye generated is related to the presence or amount of analyte or target ligand in a sample; such enzymes can be conjugated to antibodies or ligand analogues. Fluorescent dyes can also be conjugated to antibodies or ligand analogues, and the amount of fluorescent signal is measured as a result of the assay process to define the analyte presence or amount.
An objective with immunoassays is to make them more sensitive, so that a lower concentration of analyte can be measured. To increase sensitivity, one can conjugate a biological reagent to a fluorescent particle. As used herein, “fluorescent particles” can be formed of a synthetic material such as latex, can comprise a natural material such as liposomes, or combinations of natural and synthetic materials. In the preparation of the fluorescent particles, the fluorescent dyes or enzymes are generally imbibed into and/or adsorbed onto the particles, using mixed organic and aqueous solutions. The fluorescent dyes or enzymes incorporated into particles are usually water insoluble, whereas the dyes or enzymes conjugated directly to biological reagents are generally water soluble.
One can conjugate, for example, a biological reagent to a fluorescent particle that contains many fluorescent molecules or enzymes capable of converting a substrate into a fluorescent product. In the case of conjugating an antibody to a fluorescent particle, the analyte that is bound by the antibody conjugated to the fluorescent particle containing many fluorescent molecules is thus associated with a larger signal as compared with binding an antibody that is conjugated with a single fluorescent molecule or enzyme.
Examples of immunoassays that incorporate fluorescent dyes or enzymes are numerous and some are described in U.S. Pat. Nos. 4,283,382; 4,351,760; 4,420,568; 4,868,132; 4,876,190; 5,075,215; and, E.P.O. Application Number PCT/US87/03226.
Immunoassays are characterized as either competitive or non-competitive. Competitive immunoassays generally operate by a competition between a ligand analogue signal generator complex and a ligand (i.e., the analyte) for binding to a limited amount of antibody. Competitive immunoassays can also function by a competition between a protein or an antibody signal generator conjugate and an analyte which is a protein or an antibody, respectively, for binding to a limited amount of ligand. Non-competitive immunoassays generally function by having two different antibodies bind the analyte at different epitopes, one antibody conjugated to the signal generator and the other antibody in solution or bound to a solid phase in order to facilitate separation of unbound signal generator from the bound signal generator.
The antibody concentration in an immunoassay generally dictates the kinetics of the immunoassay, as well as the fraction of analyte bound to the antibody. In general, to accelerate the binding of an analyte to an antibody so that the reaction achieves a desired reaction rate or equilibrium in a desired time, the highest antibody concentration that is feasible is used in the assay; this is particularly true for non-competitive immunoassays. Thus, a high antibody concentration is often employed in fluorescent assays. Generally a high antibody concentration is also associated with a high fluorescent dye concentration, since each make up the antibody:particle conjugate or the antibody:dye conjugate.
Thus, for immunoassays, fluorescent particles are often used to obtain a more sensitive measure of analyte concentration. The quantity of a fluorescent molecule that needs to be associated with a complex of an analyte and a fluorescent conjugate is generally determined based on the sensitivity requirements of the assay. Thus, for sensitive assays, the amount of fluorescent dye or enzyme associated with the analyte in the assay process, is maximized. In such a case, the local concentration of fluorescent molecules is very high in the immediate vicinity of the biological reagent because they are in close proximity to each other.
Nucleic acid assays also utilize fluorescent sinal generators. Signal generators associated with nucleic acids are used in assays to measure nucleic acids complementary with the nucleic acid associated with a signal generator. In order to optimize the hybridization kinetics with the target nucleic acid and also to achieve the most sensitive assay, these nucleic acids are also at a high local concentration in the vicinity of fluorescent molecules. To create a nucleic acid conjugate, a signal development element is conjugated to a nucleic acid sequence. The signal development element can be a fluorescent particle or a fluorescent molecule.
Previously, however, a problem was encountered with fluorescent conjugates for use in assays. Often, fluorescent conjugates were not stable. The reactivity of such conjugates was found to decay over time, such that the function or properties of a biological reagent in a fluorescent conjugate would change with time. For example, the binding affinity of an antibody linked to a fluorescent signal would often decrease with time. The mechanism(s) for this degradation of the properties of biological reagents in fluorescent conjugates was not known. It was presumed, however, that the degradation occurred consequent to the release or uncoupling of the biological reagent from the fluorescent molecule.